Planarian flatworms are famous for their ability to rapidly regenerate new heads or even entire organisms from a tiny fragment of the animal. Planarian regeneration involves a population of proliferative cells (neoblasts) that include pluripotent adult stem cells (cNeoblasts) that can produce every cell of the adult animal. Despite centuries of fascination with regeneration, mechanistic explanations await elucidation. The broad, long-term objectives of this proposal are to use planarians as a model system to identify and understand the molecular mechanisms that regulate stem cells to promote regeneration. The specific aims are: 1) to determine the functions of neoblast regulatory genes, 2) to identify the cellular basis for regeneration, and 3) to identify wound-induced mechanisms that activate neoblast regenerative responses. Stem cells and regenerative biology are the subjects of recent and intense interest for regenerative medicine. In addition, the misregulation of stem cells may be central to many types of cancer. A newly developed arsenal of tools for molecular genetic study of planarians now exists. For example, systematic gene perturbation with RNA interference (RNAi) is now possible and the planarian genome has been sequenced. Greater than half of planarian genes have counterparts in the human genome; therefore, planarian studies should identify conserved stem cell regulatory genes. Aim #1 will determine the role of the transcription factor SoxP-1 in regulating gene expression in the neoblasts, utilizing RNAi, flow cytometry, and quantitative mRNA sequencing (RNA-seq). cNeoblasts can produce colonies of descendant cells including neoblasts and differentiating cells spanning germ layers. Aim #1 will utilize features of cNeoblast colonies to characterize roles for genes with enriched expression in the neoblast population. Aim #2 will utilize RNA seq and in situ hybridizations to identify the molecular features of the cNeoblast that distinguish it from lineage-committed descendant cells. Aim #2 will also utilize cNeoblast isolation and transplantation to develop neoblast genetic manipulation tools. Aim #3 will determine how organ precursors respond to injuries that remove target tissues. Specifically, eyes will be injured partially or completely to investigate whether eye precursors are induced by small eye injuries and in appropriate numbers tailored to the injury type. Finally, the roles of genes induced to be expressed in neoblasts following injury, including two genes encoding conserved Runx-family transcription factors, will be determined using RNAi and cellular assays for neoblast response to wounds. Successful completion of proposed aims will greatly advance our understanding of the mechanistic basis for regeneration and advance planarians as a model system for the study of genes conserved in humans in stem cell and regenerative biology, areas of great importance in human health.